Monoclonal antibody ds6, tumor-associated antigen ca6, and methods of use thereof

ABSTRACT

The present application describes a monoclonal antibody selected from the group consisting of monoclonal antibody DS6, monoclonal antibodies that specifically bind to the antigen or epitope bound by monoclonal antibody DS6, and fragments of the foregoing that specifically bind to the antigen or epitope bound by monoclonal antibody DS6. Methods of use of such antibodies and the isolated antigen bound by such antibodies are also described.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/287,633, filed Nov. 2, 2011, now issued U.S.Pat. No. 8,728,741, which is a continuation application of U.S. patentapplication Ser. No. 12/363,191, filed Jan. 30, 2009, now issued U.S.Pat. No. 8,067,186, which is a continuation application of U.S. patentapplication Ser. No. 11/680,190, filed Feb. 28, 2007, now issued U.S.Pat. No. 7,507,410, which is a divisional application of U.S. patentapplication Ser. No. 10/465,176, filed Jun. 19, 2003, now issued U.S.Pat. No. 7,351,805, which is a divisional application of U.S. patentapplication Ser. No. 09/641,499, filed Aug. 18, 2000, now issued U.S.Pat. No. 6,596,503. The disclosures of each of these applications are tobe incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention concerns the monoclonal antibody DS6,tumor-associated antigen CA6, and methods of use thereof.

BACKGROUND OF THE INVENTION

Malignancy-associated changes of tumor cell membrane sialoglycoproteinsand sialoglycolipids (gangliosides) have been observed for decades andare implicated in many aspects of the transformed phenotype, includingaltered adhesive properties and metastatic potential (Bhavanandan, V.P., Furukawa, K., In: A. Rosenberg (ed.), Biology of the Sialic Acids,pp. 144-196, Plenum Press, New York (1995)). Altered, re-expressed orover-expressed sialoglycoconjugates, viewed as tumor-associated antigens(TAAs), are being investigated as targets for a variety ofantibody-directed cancer therapies. Some examples include monoclonalantibodies (mabs) directed against sTn (TAG-72) in breast cancer therapy(Estava, F. J. and Hayes, D. F. Monoclonal antibody-based therapy ofbreast cancer. In: M. L. Grossbard (ed.), Monoclonal antibody-basedtherapy of cancer, pp. 309-338, Marcel Dekker, New York (1998)) and mabsto gangliosides such as GD2, GD3 and GM2 in the therapy of lung cancerand melanoma (Butler, M. O. and Haluska, F. G., In: M. L. Grossbard(ed.), Monoclonal antibody-based therapy of cancer, pp. 339-364, MarcelDekker, New York (1998); Grant, S. C. et al., In: M. L. Grossbard (ed.),Monoclonal antibody-based therapy of cancer, pp. 365-395, Marcel Dekker,New York (1998)). Nevertheless, there remains a need for new ways todetect and treat cancer in patients.

SUMMARY OF THE INVENTION

The present invention is based upon the development of murine monoclonalantibody DS6 deposited in the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va., 20110-2209, on Jun. 11, 2002 anddesignated PTA-4449. This antibody immunohistochemically reacts with anantigen, CA6, which is expressed by human serous ovarian carcinomas butnot expressed by normal ovarian surface epithelium or mesothelium. TheCA6 antigen has a limited distribution in normal adult tissues and ismost characteristically detected in fallopian tube epithelium, innerurothelium and type 2 pneumocytes. Pretreatment of tissue sections witheither periodic acid or neuraminidase from Vibrio cholerae abolishesimmunoreactivity with DS6 indicating that CA6 is aneuraminidase-sensitive and periodic acid-sensitive sialic acidglycoconjugate (“sialoglycotope”). SDS-polyacrylamide gelelectrophoresis of OVCAR5 cell lysates reveals the DS6 epitope to beexpressed on an 80 kiloDalton nondisulfide-linked glycoproteincontaining N-linked oligosaccharides. Two-dimensional nonequilibrium pHgradient electrophoresis gels indicates an isoelectric point ofapproximately 6.2-6.5. DS6 immunostaining can be partially diminished bypretreatment of tissue sections with chloroform/methanol, suggestingthat DS6 may also be expressed as a glycolipid. Comparison of theimmunohistochemical distribution of the CA6 antigen in human serousovarian adenocarcinomas reveals similarities to that of CA125, however,distinct differences and some complementarity of antigen expression arerevealed by double-label, two-color immunohistochemical studies. TheDS6-detected CA6 antigen appears distinct from other well-characterizedtumor-associated antigens, including MUC1, CA125, and the histo-bloodgroup-related antigens, sLea, sLex and sTn.

A first aspect of the present invention is a monoclonal antibodyselected from the group consisting of monoclonal antibody DS6 andmonoclonal antibodies that specifically bind to the antigen or epitopebound by monoclonal antibody DS6, and fragments of the foregoing thatspecifically bind to the antigen or epitope bound by monoclonal antibodyDS6.

A second aspect of the present invention is a method of screening forthe presence of cancer in a human subject, comprising the steps of: (a)contacting a biological sample taken from said subject with an antibodyas described above under conditions permitting said antibody tospecifically bind an antigen in the sample to form an antibody-antigencomplex; and then (b) determining the amount of antibody-antigen complexin the sample as a measure of the amount of antigen in the sample,wherein an elevated level of the antigen in the sample is associatedwith the presence of cancer in said subject.

A further aspect of the present invention is a method of treating cancerin a subject in need thereof, comprising, administering to a subjectafflicted with cancer a monoclonal antibody as described above in atherapeutically effective amount.

A still further aspect of the present invention is isolatedtumor-associated antigen CA6, an about 80 kDa N-linked glycoprotein,reduced or non-reduced, with a PI of about 6.2-6.5, and containing asialiadase and periodate sensitive epitope called DS6. Such antigen maybe isolated by affinity purification with a monoclonal antibody DS6 asdescribed herein.

The foregoing and other objects and aspects of the present invention areexplained in detail in the drawings herein and the specification setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: DS6 immunohistochemical staining pattern on normal humantissues, A-D, with AEC (red) as the chromagen, hematoxylin counterstain:(A) Fallopian tube with apical staining of cells lining lumen, (B)apical aspect of type 2 pneumocytes of lung, (C) transitional epitheliumof ureter with DS6 staining of luminal-facing aspects of inner celllayers, (D) squamous metaplasia of uterine cervix with rim patternoutlining cell membrane.

FIG. 2: (A) DS6 immunoperoxidase buffer control, i.e., withoutneuraminidase, on serous ovarian carcinoma, (B) DS6 immunoperoxidase onserous ovarian carcinoma following neuraminidase (Vibrio cholerae)treatment. C-F: Double label, sequential two color immunohistochamicalstaining of serous ovarian carcinomas with DS6 and OC125, hematoxylincounterstain: (C) DS6 (DAB, brown) followed by mouse Ig control (VIP,purple) shows a typical staining pattern for DS6 on tumors with bothluminal and cytoplasmic staining, (D) OC125 (DAB, brown) followed bymouse Ig control (VIP, purple) shows OC125 with a luminal pattern and afocus demonstrating a rim pattern along tumor cell membranes, (E) DS6(DAB) staining followed by OC125 (VIP) on a tumor demonstrating adiscreet area of staining (purple chromagen) that is DS6-nonreactive butOC125-reactive, (F) OC125 (DAB) staining followed by DS6 (VIP) stainingreveal areas of DS6-reactivity that were not detected by OC125.

FIG. 3: (A) Two-dimensional nonequillibrium pH gradient gelelectrophoresis (NEPHGE) analysis of the DS6-detected CA6 antigen.Samples were analyzed on NEPHGE gels (pI range˜3.2-7.8) in the firstdimension, followed by 13% SDS-PAGE gels in the second dimension, allunder reducing conditions. (B) One dimensional SDS-PAGE analysis on 13%gels under reducing conditions and in (C) on 10% gels under nonreducingconditions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Any type of antibody may be used in the present invention. The term“antibodies” as used herein refers to all types of immunoglobulins,including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG areparticularly preferred. The antibodies may be monoclonal or polyclonal(with monoclonal antibodies preferred) and may be of any species oforigin, including (for example) mouse, rat, rabbit, horse, or human.See, e.g., M. Walker et al., Molec. Immunol. 26, 403-11 (1989). Antibodyfragments that retain specific binding to the protein or epitope boundby DS6 are included within the scope of the term “antibody” and include,for example, Fab, F(ab′)2, and Fc fragments, and the correspondingfragments obtained from antibodies other than IgG. Such fragments can beproduced by known techniques. The antibodies may be chimeric orhumanized, particularly when they are used for therapeutic purposes.

Subjects or patients with whom the instant invention is concerned areprimarily human subjects, but the invention may also be employed withother mammalian subjects such as dogs, cats, and horses for veterinarypurposes. Subjects may be male or female.

Applicants specifically intend that all United States patent referencescited herein be incorporated herein by reference in their entirety.

1. Antibody Production.

Monoclonal antibodies of the present invention may be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120). Briefly, the procedure is asfollows: an animal is immunized with antigen or immunogenic fragments orconjugates thereof. For example, haptenic oligopeptides of antigen canbe conjugated to a carrier protein to be used as an immunogen. Lymphoidcells (e.g. splenic lymphocytes) are then obtained from the immunizedanimal and fused with immortalizing cells (e.g. myeloma orheteromyeloma) to produce hybrid cells. The hybrid cells are screened toidentify those that produce the desired antibody.

Human hybridomas, which secrete human antibody, can be produced by theKohler and Milstein technique. Although human antibodies are especiallypreferred for treatment of human, in general, the generation of stablehuman-human hybridomas for long-term production of human monoclonalantibody can be difficult. Hybridoma production in rodents, especiallymouse, is a very well established procedure and thus, stable murinehybridomas provide an unlimited source of antibody of selectcharacteristics. As an alternative to human antibodies, the mouseantibodies can be converted to chimeric murine/human antibodies bygenetic engineering techniques. See V. T. Oi et al., Bio Techniques4(4):214-221 (1986); L. K. Sun et al., Hybridoma 5 (1986).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (S. L. Morrison, et al. Proc. Natl.Acad. Sci. 81, 6851-6855 (1984); M. S. Neuberger et al., Nature312:604-608 (1984); S. Takeda, S. et al., Nature 314:452-454 (1985)).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceCA6-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (D. R.Burton, Proc. Natl. Acad. Sci. 88, 11120-3 (1991)).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature (R.Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837 (1989)); G. Winteret al., Nature 349, 293-299 (1991)).

Polyclonal antibodies used to carry out the present invention may beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withthe antigen to which monoclonal antibody DS6 binds, collecting immuneserum from the animal, and separating the polyclonal antibodies from theimmune serum, in accordance with known procedures. Depending on the hostspecies, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable.

Antibodies that bind to the same epitope (i.e., the specific bindingsite) that is bound by the DS6 antibody can be identified in accordancewith known techniques, such as their ability to compete with labeledantibody to in binding to CA6 in a competitive binding assay.

Monoclonal antibodies specific for CA6 epitope can be used to produceanti-idiotypic (paratope-specific) antibodies. See e.g., McNamara etal., Science 220, 1325-26 (1984), R. C. Kennedy, et al., Science 232,220 (1986). These antibodies resemble the CA6 epitope and thus can beused as an antigen to stimulate an immune response against CA6, or toscreen other antibodies for the ability to specifically bind to the sameepitope bound by monoclonal antibody DS6.

DS6 can also be bound to a column (such as Protein A/G) and used toobtain purified CA6 antigen from a variety of sources, including humantissues/tumors and cancer cell lines that produce CA6. Such purified CA6antigen can then be used to produce additional antibodies (monoclonaland/or polyclonal) by methods described above. Some of these antibodiesmay react with the DS6 epitope while others can recognize differentepitopes on CA6. In one example, enzyme immunoassay to detect antigensin human body fluids often use a combination of antibodies thatrecognize different, non-sterically interfering epitopes on the sameantigen. For this purpose, a column containing immobilized neuraminidasecould be used to desialylate purified CA6; the desialylated CA6 can thenbe used as an immunogen to produce antibodies (monoclonal and/orpolyclonal) that react with non-DS6 epitopes on CA6. These antibodiescould then be used as either capture and/or tracer antibodies in anenzyme immunoassay for quantitation of the CA6 antigen for use inmonitoring of CA6 in pathologic states.

2. Immunoassay Techniques.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof, which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980)(CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for ModulatingLigand-Receptor Interactions and their Application,” U.S. Pat. No.4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat.No. 4,376,110 to David et al., titled “Immunometric Assays UsingMonoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled“Macromolecular Environment Control in Specific Receptor Assays,” U.S.Pat. No. 4,233,402 to Maggio et al., titled “Reagents and MethodEmploying Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al.,titled “Heterogenous Specific Binding Assay Employing a Coenzyme asLabel.”

Antibodies as described herein may be coupled or conjugated to a solidsupport suitable for a diagnostic assay (e.g., beads, plates, slides orwells formed from materials such as latex or polystyrene) in accordancewith known techniques, such as precipitation.

Antibodies as described herein may likewise be conjugated to detectablegroups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g.,horseradish peroxidase, alkaline phosphatase), fluorescent labels (e.g.,fluorescein), chemiluminescent labels (e.g., acridinium groups,metalloporphyrins such as phthalocyanine dyes, luminol, etc.), metalatoms (e.g., technetium-99m), etc., in accordance with known techniques.See, e.g., U.S. Pat. No. 4,472,509 to Gansow (metal chelates tomonoclonal antibodies); U.S. Pat. No. 5,061,641 to Schochat et al.; andU.S. Pat. No. 4,861,869 to Nicoleotti et al. (radiolabelling proteins).

Immunoassays, or other types of assays to detect and/or quantitate thelevel of the CA6 antigen in samples as described below, may be used inscreening assays to detect pathologic states associated with aberrantlevels of CA6 expression (e.g., tumors, inflammatory states), diagnosticstudies, prognostic studies, or to monitor the progression or diminutionof CA6 expression in correlation with disease state.

Samples that may be collected for use in carrying out the immunoassaymay be tissue samples from the organ or tissue of interest within thesubject, such tissue generally of most interest being those types oftissues/cells that express differing amounts of CA6 in pathologic statesas compared to non-pathologic states, or biological fluids such as blood(including blood fractions such as blood plasma or blood serum), urine,cerebrospinal fluid, etc). Examples may include overexpression oraberrant expression of CA6 in various types of malignancies as will beseen in the Tables below (e.g. ovarian cancer, endometrial cancer,pancreatic cancer, breast cancer, urinary bladder cancer, lung cancer,etc.), as well as overexpression or aberrant expression in otherpathologic states, such as overexpression of CA6 by pneumocytes in lungsdisease, for example, pneumonia.

A biological sample may be a cell sample, with an intervening culturingstep being performed between the time the cell sample is collected fromthe subject and the immunoassay is carried out on the biological sample.

For immunohistological techniques, a tissue sample is collected from thesubject, and the presence or absence of binding of an antibody of theinvention is detected. The presence of binding of the antibody in anabnormal pattern or a pattern indicative of a tumor or cancer indicatesthe presence of a tumor or cancer in the subject from which the tissuesample is collected. The presence of the antigen in a metastatic tumordeposit can also be used to determine a likely source of the primarytumor. Any suitable immunohistology format may be used. The tissuesample may include patient biopsies, resections or cells for cytologicstudy. A similar technique to immumohistogy is the use of similartechniques to detect and/or phenotype cells in body fluids or othersuspensions as is used for flow cytometric examination.

For in vivo diagnostic purposes the antibody according to the inventionis coupled to or provided with a suitable externally detectable label,such as e.g. a radiolabel as described above or a metal atom (e.g.,technetium-99m), and administered to a subject (e.g., by intraveneous orintraarterial injection), in an amount sufficient to produce anexternally detectable signal, whereupon the possible localizedaccumulation of antibody in the body is determined, with a localizedaccumulation of the antibody (in a region other than that which wouldordinarily be expected for normal subjects or subjects free of disease)indicating the present of a tumor in that subject.

3. Therapeutic Antibodies and Methods.

Monoclonal antibodies used for therapy (i.e., in a method of combatingcancer) may be monoclonal antibodies per se or monoclonal antibodiescoupled to a therapeutic agent. Such antibodies are referred to hereinas therapeutic monoclonal antibodies. Any therapeutic agentconventionally coupled to a monoclonal antibody may be employed,including (but not limited to) radioisotopes, cytotoxic agents, andchemotherapeutic agents (See generally Monoclonal Antibodies and CancerTherapy (R. Reisfeld and S. Sell Eds. 1985)(Alan R. Liss Inc. NY); U.S.Pat. No. 5,558,852 to Bigner and Zalutsky; U.S. Pat. No. 5,624,659 toBigner and Zalutsky).

Therapeutic agents may be conjugated or coupled to the antibody bydirect means or indirect means (e.g., via a chelator); such as theIodogen method or with N-succinimidyl-3-(tri-n-butylstanyl)benzoate (the“ATE method”), as will be apparent to those skilled in the art. See,e.g., M. Zalutsky and A. Narula, Appl. Radiat. Isot. 38, 1051 (1987).

Examples of radioisotopes, which may be coupled to a therapeuticmonoclonal antibody, include, but are not limited to, ¹³¹I, ⁹⁰Y, ²¹¹At,²¹²Bi, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, and ²¹²Pb. Examples of chemotherapeuticagents, which may be coupled to a therapeutic monoclonal antibody,include, but are not limited to, methotrexate. Examples of cytotoxicagents, which may be coupled to a therapeutic monoclonal antibody,include, but are not limited to, ricin (or more particularly the ricin Achain).

The monoclonal antibodies of the invention can be conjugated to and usedas targeting agents for genes (immunogenes, suicide genes),immunoliposomes, boron neutron capture therapy, photo sensitizers forphotodynamic therapy, and other types of therapies that can be directedby antibodies.

It will be appreciated that monoclonal antibodies per se which are usedas therapeutic monoclonal antibodies incorporate those portions of theconstant region of an antibody necessary to evoke a therapeuticallyuseful immunological response in the subject being treated.

Therapeutic monoclonal antibodies may be provided in lyophylized form ina sterile aseptic container or may be provided in a pharmaceuticalformulation in combination with a pharmaceutically acceptable carrier,such as sterile pyrogen-free water or sterile pyrogen-free physiologicalsaline solution.

The therapeutic methods disclosed herein may be employed with subjectssuspected of having a variety of tumors, whether primary or metastaticor micrometastatic (see Tables below), of particular importance aretumors of the ovary, endometrium, breast, urinary bladder, pancreas andlung. DS6 may also be of therapeutic use in other types of neoplasms,especially if used as part of a panel or combination of therapeuticantibodies, each with different specificities (Smith, N. L. et al, HumanAntibodies, 9, 61-65, (1999); Oldham, R. K., Mol Biother., 3, 148-161,(1991)).

For administration, the antibody will generally be mixed, prior toadministration, with a non-toxic, pharmaceutically acceptable carriersubstance (e.g. normal saline or phosphate-buffered saline), and may beadministered using any medically appropriate procedure, e.g.,intravenous or intra-arterial administration, injection into thecerebrospinal fluid). In certain cases, intradermal, intracavity,intrathecal or direct administration to the tumor or to an arterysupplying the tumor is advantageous.

Dosage of the antibody will depend, among other things, on the tumorbeing treated, the route of administration, the nature of thetherapeutic agent employed, and the sensitivity of the tumor to theparticular therapeutic agent. For example, the dosage will typically beabout 1 to 10 micrograms per Kilogram subject body weight. In anotherexample, where the therapeutic agent is ¹³¹I, the dosage to the patientwill typically be from 10 mCi to 100, 300 or even 500 mCi. Statedotherwise, where the therapeutic agent is ¹³¹I, the dosage to thepatient will typically be from 5,000 Rads to 100,000 Rads (preferably atleast 13,000 Rads, or even at least 50,000 Rads). Doses for otherradionuclides are typically selected so that the tumoricidal dose willbe equivalent to the foregoing range for ¹³¹I. The antibody can beadministered to the subject in a series of more than one administration,and regular periodic administration will sometimes be required.

The present invention is explained in greater detail in the followingnon-limiting Examples. Abbreviations used: mab (monoclonal antibody),TAA (tumor-associated antigen), IH(immunohistochemistry/immunohistochemical), kiloDalton (kDa).

Example 1 Monoclonal Antibody DS6 Detects a Tumor-AssociatedSialoglycotope Expressed on Human Serous Ovarian Carcinomas

We have recently developed a murine monoclonal antibody (mab), cloneDS6, using human serous ovarian carcinoma as the immunogen (Smith, N. L.et al., Human Antibodies, 9, 61-65, (1999); Kearse, K. P. et al, Int. J.Cancer, in press, (2000)). We now report the immunohistochemical (IH)distribution of the DS6-detected antigen, CA6, in normal adult humantissues and selected neoplasms and characterize the epitope as asialoglycotope.

1. Materials and Methods

Hybridoma Production.

Immunizations, fusions and screening were performed essentially asdescribed previously using P3X63-Ag8.653 myeloma cells with human serousovarian carcinoma as the immunogen (Wennerberg, A. E. et al., Am. J.Pathol., 143(4), 1050-1054 (1993)). Institutional Animal Care and Useapproval was obtained. Preliminary and secondary screenings were byavidin-biotin IH (mouse IgG peroxidase kit, Vector Laboratories,Burlingame, Calif.) on tissue sections of ovarian carcinomas andselected normal adult tissues. DS6 was chosen for further studies and,after several rounds of single cell cloning by limiting dilution, wasisotyped as an IgG1 (ImmunoType Kit, Sigma Chemicals, St. Louis, Mo.).Supernatant was collected in batch for the studies in this report fromDS6 cells grown in DMEM-F12 supplemented with 10% horse serum.Quantitation of murine. IgG1 in the supernatant was by EIA methodology(performed by the East Carolina University Hybridoma Core Facility,Greenville, N.C.).

Immunohistochemistry.

Tissue culture supernatant (40 ug DS6/ml) was used for IH as above withAEC (Vector Laboratories) as the chromagen. Frozen cryostat tissuesections were air-dried, acetone-permeabilized while formalin-fixed,paraffin-embedded tissue sections were de-waxed through solvents andrehydrated, prior to blocking of endogenous peroxidase withmethanol/hydrogen peroxide and subsequent immunostaining. Mouse IgG1 wasused as a negative control mab. Anti-transferring receptor mab (IgG1)was used as a positive control on frozen cryostat sections whileanti-smooth muscle myosin mab (IgG1) was used as a positive control onformalin-fixed tissues (all control antibodies from DAKO, Carpenteria,Calif.).

Human Adult Tissues.

Samples of grossly normal, incidental tissues were obtained fresh fromeither autopsy or the surgical bench from individuals withoutsignificant pathology in the primary organ or tissue; half were used forair-dried, acetone-permeabilized cryostat sections and half were fixedin buffered formalin and paraffin-embedded. Additional normal tissues aswell as a pilot selection of gynecologic and related neoplasms wereobtained from the archival tissue stores of formalin-fixed,paraffin-embedded tissue blocks of the University hospital (Table 1 andTable 2). Fresh specimens of serous ovarian tumors and normal fallopiantubes were obtained from surgical cases and cryostat sections were cutfor chloroform extraction studies. Use of incidental human tissues hadprior approval of the Institutional Review Board.

Following completion of the normal tissue distribution and pilot studyon gynecologic tumors, an expanded study was undertaken. 296formalin-fixed, paraffin-embedded human gynecologic neoplasms wereimmunohistochemically examined using DS6 as the primary antibody (Table3). A large study of non-gynecologic neoplasms followed (n=approx 1200)as seen in Tables 4-8.

Pretreated and/or Preabsorbed Immunohistochemical Studies.

For periodic oxidation of carbohydrates, formalin-fixed,paraffin-embedded sections of normal human stomach, small bowel, colon,fallopian tube, breast and serous ovarian carcinoma were pretreated,prior to IH, with 20 mM periodic acid in 0.5M acetate buffer, pH 5.0 asdescribed by Cao (Cao, Y. et al., J. Histochem. & Cytochem., 45(11),1547-1557 (1997); Cao, Y. et al., Tumor Biol. 19(1), 88-99 (1998).).Parallel control sections were incubated in acetate buffer withoutperiodic acid to control for loss of reactivity due to pH or buffer. Fordetection of sialic acid, 0.02 U/ml neuraminidase from Vibrio cholerae(reacts with sialic acids in α 2-3,-6,-8 linkages, BoehringerMannheim/Roche, Indianapolis, Ind.), in 0.01M Ca++ in PBS was used topretreat formalin-fixed tissue sections of the above tissues withparallel control sections pretreated with Ca++/PBS buffer withoutneuraminidase. An additional control for the adequacy of sialic acidremoval (via unmasking of PNA lectin-binding sites) was the inclusion ofa section of formalin-fixed kidney, run as above, but stained withPNA-biotin (EY Labs, San Mateo, Calif.) in place of the DS6 primary andbiotin-labeled secondary antibodies. For removal of glycolipids prior toDS6 immunostaining, unfixed, air-dried cryostat sections of normalfallopian tubes and serous ovarian tumors were placed in 5% chloroformin methanol for 10 minute (Zhang, S. et al., Int. J. Cancer, 73, 50-56(1997)).

To determine if DS6 reacts with several commercially available, highlypurified carbohydrate antigens (sTn, Tn, Tf, sLea, Calbiochem, La Jolla,Calif.), the antigens were used to preabsorb DS6 prior to immunostaining(Taylor, C. R., In: C. R. Taylor (ed.), Immunomicroscopy: A diagnostictool for the surgical pathologist, pp. 23-43, W.B. Saunders,Philadelphia (1986); Elias, J. M., Immunohistopathology: a practicalapproach to diagnosis, pp. 53-56, American Society of ClinicalPathologists, USA (1990)). Briefly, carbohydrate antigens were added toDS6 supernatant at 10 ug, 50 ug, 250 ug and 500 ug carbohydrate to 4 ugDS6 and allowed to react at 4° C. overnight. This preabsorbedsupernatant was then used by IH to study sections of formalin-fixed,paraffin-embedded serous ovarian carcinomas and compared to controlsections stained with unabsorbed DS6 supernatant.

Double-Immunolabel, Two-Color IH on Tissue Sections.

Small sections (up to 1.0 cm) of eight formalin-fixed andparaffin-embedded serous adenocarcinomas and a metastasis of each werere-embedded into two paraffin blocks for single antibody and doublelabel/sequential IH (Battifora, H., Lab. Invest., 55, 244-248 (1986)).Seven of the cases of adenocarcinoma had their primary site in theovary, and one case was a primary papillary serous carcinoma of theperitoneum. A section of normal fallopian tube was also included in eachblock. A manufacturer's protocol (Vector Laboratories) for two-color,double-label IH was used: in brief, one of the murine mabs is used in astandard avidin-biotin IH technique with DAB (brown product, VectorLaboratories) as chromagen and then the entire process is repeated onthe same slide using the alternate murine mab with VIP (“Very IntensePurple”; purple product, Vector Laboratories) as the chromagen. DS6(neat) supernatant was used as one mab and a murine anti-Ca125 (OCH125Level I, an IgG1, Signet Laboratories, Dedham, Mass.) as the alternativemab. DAB was consistently used as the first chromagen and VIP as thesecond chromagen in the sequence. For double-label studies with both DS6and OC125, the order of the primary antibodies was switched whilemaintaining the order of the chromagens so that each mab was evaluatedboth as a first and second mab in the sequence. Controls consisted of aprimary mab (DS6 or OC125) with DAB followed by a mouse ascites negativecontrol (Signet Laboratories) as the second mab with VIP. Antigenretrieval (10 mM, pH 6.0 citrate buffer) by microwave proceeded alldouble-label staining to assure antigen detection by mab OC125.

Immunoblotting and SDS-PAGE Gel Electrophoresis.

Lysates of OVCAR5, a human ovarian carcinoma cell line (a generous giftfrom Dr. Thomas Hamilton, Fox Chase Cancer Center, Philadelphia, Pa.)that is reactive with DS6 by IH, were analyzed on one andtwo-dimensional SDS-polyacrylamide gel electrophoresis as previouslydescribed (Van Leeuwen, J. E. M. and Kearse, K. P., J. Biol. Chem., 271,25345-25349 (1996)). Gels were transferred to a nitrocellulose membraneand probed with DS6 or control antisera diluted in PBS containing 5%powdered milk, washed, and incubated with protein A-HRP (ICN, Irvine,Calif.) in PBS (5% milk). Protein A-HRP complexes were visualized bychemiluminescence performed according to manufacturer's instructions(Pierce Chemical Company, Rockford, Ill.). N-linked glycans were removedfrom precipitated proteins using an Enzymatic Deglycosylation kit(Glyko, Inc., Novato, Calif., USA).

2. Results

Distribution of CA6 in Normal Tissues and Tumors by IH.

The results of IH staining using DS6 on formalin-fixed,paraffin-embedded tissues are similar to those obtained onacetone-permeabilized cryostat sections, consistent with an epitope thatis fairly resistant to the effects of formalin-fixation. The IH patternof DS6 on cells of normal adult tissues is predominately an apical,epithelial cell membrane localization in cells lining certain tubular orsaccular structures (e.g., fallopian tube, pulmonary alveoli) and alongthe luminal-facing aspect of the inner layers of some stratifiedepithelia, such as urothelium (FIG. 1, A-C). In a few instances, such asmetaplastic cervical epithelium and squamous metaplasia of amnion, theentire cell membrane can be decorated giving a rim pattern to the cell(FIG. 1, D). There is absent to minimal cytoplasmic expression of theantigen in any of the normal human adult epithelia studied.

The immunohistochemical distribution of the CA6 antigen in various typesof normal adult tissues in given in Table 1. In normal adult tissues,the CA6 antigen is most consistently detected in fallopian tubeepithelium, urothelium and type 2 pneumocytes. In other tissues thathave DS6 immunoreactivity, the CA6 antigen has a more inhomogeneous andvariable expression pattern. This is particularly evident in ductalstructures, where expression can vary from one duct cross section toanother within a given histologic preparation.

In a pilot study of selected gynecologic neoplasms (Tables 2), CA6 isexpressed along the apical membranes of many cells in ovarian serouscystadenomas. In ovarian serous adenocarcinomas, the level of CA6expression is more intense and is additionally seen in the cytoplasm ofmany malignant cells and extracellularly within gland lumen (FIG. 2, C).A similar luminal/apical pattern with variable cytoplasmic staining isalso seen in the other adenocarcinomas that are DS6 immunoreactive.Mucinous ovarian tumors are not characteristically DS6 immunoreactive asno reactivity is seen in benign and borderline mucinous tumors and isonly seen quite focally in 3 of 9 mucinous adenocarcinomas. CA6 is notdetected in normal or hyperplastic mesothelium and does not appear to becharacteristic of mesotheliomas (1/6), but is readily detected in seroussurface carcinomas of the peritoneum (3/3).

Sensitivity of CA6 Epitope to Periodic Acid, Neuraminidase, andChloroform Extraction.

To characterize the CA6 epitope, sections of DS6-reactive (IH) fallopiantube and serous ovarian carcinoma, as well as DS6 nonreactive stomach,small bowel, colon and breast, were subjected to a series ofpretreatments with either periodic acid or neuraminidase (V. cholera)prior to immuonostaining with DS6. Pretreatment of the tissue sectionswith either periodic acid or neuraminidase (sialidase) prior to IH withDS6 results in complete abolishment of DS6 immunoreactivity in thesections of fallopian tube and ovarian carcinoma and no DS6immunoreactivity was unmasked in any of the sections of gastrointestinaltissues or breast. DS6 reactivity remained unaffected in the buffercontrol sections (FIG. 2, A-B). Preabsorption of DS6 supernatant withcommercially available purified carbohydrate antigens (sTn, sLea, Tf,Tn) under conditions of antigen excess failed to reduce the intensity ofDS6 immunostaining of serous ovarian carcinomas (data not shown).

Chloroform extraction studies were performed on sections of fallopiantubes (2) and serous ovarian tumors (3) prior to immunostaining withDS6. Control cryostat sections of fallopian tubes and ovarian tumorsshow strong staining with DS6 in the formalin-fixed as well as in theacetone-permeabilized slides. The chloroform-pretreated slides, stainedwith DS6, reveal mild to moderate reduction in staining intensity infallopian tubes and ovarian tumors respectively.

Immunoblotting and SDS-Polyacrylamide Gel Electrophoresis.

OVCAR5, a human ovarian carcinoma cell line reactive with DS6 by IH(data not shown) was used as a reproducible source of antigen. Anti-DS6immunoblots of OVCAR5 lysates gave a predominant band at approximately80 kilodaltons (kDa), with minor species at 75 kDa, 52 kDa, and 48 kDa(FIG. 3, B). Migration is similar under both reducing and nonreducingconditions and no reactivity with control (isotype matched) antibody isobserved (FIG. 3, B-C). Analysis on two-dimensional nonequilibrium pHgradient electrophoresis (NEPHGE) gels indicates an isoelectric point ofapproximately 6.2-6.5 (FIG. 3, A). Digestion studies with PNGase Fresult in a reduction in molecular weight of the 80 kDa species toapproximately 75 kDa, consistent with the presence of one to two N-likedglycan chains (data not shown).

Double-Immunolabel, Two-Color IH of Tissue Sections.

Single antibody and double-label, two-color IH studies were performedusing mabs DS6 and OC125 to determine if CA6 and CA125 are expressed bythe same or differing tumor cell populations. Both antibodies reactedwith the luminal aspect of fallopian tubes but only OC125 reacted withthe mesothelium of the fallopian tubes. All eight primary papillaryserous adenocarcinomas and their metastases show moderate to strongpositivity for DS6 and OC125 with the exception of one primary tumorthat has only scattered focal positive cells with either mab. Comparisonof tumors stained individually with either DS6 or OC125 reveal that theyreact with roughly similar proportions of tumor cells although theintensity of staining is more pronounced with DS6. While both the CA6and CA125 antigens are expressed along the luminal membranes of theneoplastic cells in many areas and in most tumors strikingly similar IHpatterns can be found, there are some distinct differences. CA6 oftenhas an additional prominent cytoplasmic component while CA125 willoccasionally show a distinct pattern of circumferential membraneexpression on the neoplastic cells in several of the tumors, giving ahoneycomb appearance to the tumor that is not as characteristic of CA6(FIG. 2, C-D). When both DS6 and OC125 are used in sequential, two-colorIH, intensely positive dark chromagen results from overlapping, duallocalization of chromagens in many areas of the tumors. This darkchromagen can be difficult to distinguish from an intensely positivedeposition from the first mab/chromagen (brown, DAB) reaction alone.However, in most tumors, focal areas of chromagen deposition resultingfrom the second mab/chromagen (purple, VIP) reaction can be clearlyvisualized (FIG. 2, E-F). Such areas result from the second mab in thesequence reacting with an antigen that is not detected by the priorapplication of the first mab.

Following the immunohistochemical studies above, a greatly expandednumber of gynecologic neoplasms, as well as non-gynecologic neoplasms,were similarly studies for DS6 immunoreactivity. The results are listedin Tables 3-8. As can be seen, the CA6 antigen is not restricted toovarian carcinomas but can be expressed by a variety of othercarcinomas, including, but not limited to, those of the breast,endometrium, pancreas, urinary bladder and lung.

3. Discussion

As CA6 has only a limited distribution in the normal tissues studied, isnot detected in normal ovarian surface epithelium, yet is expressed inserous ovarian carcinomas which arise from the surface epithelium(18/18), it can be described as a TAA (Suresh, M. R., Anticancer Res.,16(4B), 2273-2277 (1996); Khawli, L. A. and Epstein, A. L., Q.J. Nucl.Med., 41, 25-35 (1997)). Immunohistochemical studies of DS6 on a widevariety of human carcinomas, lymphomas and sarcomas can be seen inTables 2-8 and demonstrate that DS6 is not a pan-carcinoma marker butrather is characteristic of specific types of epithelial malignancies(e.g., characteristic of serous ovarian carcinomas but seldom reactivewith colon carcinomas). Analyzing the IH distribution of CA6 in normaltissues, similarities, as well as differences, can be noted to otherreported TAAs. For instance, the pattern of reactivity in the anteriorpituitary has also been described for CA19-9 and EMA (Okubo, T. andOkabe, H., Acta Neuropathol., 93, 471-476 (1997); Pernicone, P. J. etal., In: S. S. Sternberg (ed.), Histology for pathologists, secondedition, pp 1053-1074, Lippincott-Raven, Philadelphia (1997)), thestaining of the intracellular canaliculus of parietal cells is a featureof the MUC1-related mab HMFG2 (Walker, M. M. et al., J. Clin. Pathol.,48, 832-834 (1995)), while luminal staining of ductal structures isvariably reported for a large number of TAAs, including the MUC1 mucinfamily and histo-blood group related antigens (Arklie, J. et al., Int.J. Cancer, 28, 23-29 (1981); Zhang et al., supra (1997); Cao, Y. et al.,Histochem. Cell Biol., 106, 197-207 (1996)). Reports of mabs to TAAshave been numerous and, as TAAs are not unique to tumors, it is notunexpected that some similarities in their IH distribution in normalepithelial tissues would be seen. The reported distribution ofepithelial TAAs can also vary in the hands of different investigators,perhaps reflecting differences in methodologies, grading systems,antibody preparation, tissue preparation, or fixation (Zhang et al.,supra (1997); Stein, R. et al., Int. J. Cancer, 47, 163-169 (1991);Buist, M. R. et al., J. Clin. Pathol., 48, 631-636 (1995)). We havecharacterized the IH distribution of DS6 in both cryostat andformalin-fixed, paraffin-embedded tissues to provide a range ofexperimental conditions that are in common use. Additionally, we haveclassified the types of DS6-reactive epithelial tissues as to the typesof tissues most consistently positive versus those tissues in which theexpression appears more variable. As will be discussed below, theconsistent presence or absence of CA6 in certain epithelial tissues doesappear to distinguish CA6 from certain other known TAAs. To furthercharacterize CA6, additional features of the antigen were investigated.

Pretreatment of tissue sections with either periodic acid orneuraminidase from Vibrio cholerae gives similar results. Thesetechniques are used to determine if the epitope is carbohydrate-based orsialic acid-dependent, respectively, and have also been used to unmaskhidden epitopes such as masking of the binding sites of several MUC1mabs by glycosylation (Cao et al., supra (1997); Cao et al, supra(1998)). The abolishment of DS6 immunoreactivity by both treatments isconsistent with a carbohydrate epitope that is sialic acid-dependent. NoDS6-reactive epitopes are unmasked in any of the sections by suchremoval of periodic-sensitive carbohydrates or sialic acid.

To determine if the CA6 sialoglycotope is expressed as asialoglycoprotein and/or a sialoglycolipid (ganglioside), additionalstudies were performed. SDS-PAGE immunoblots reveal that the DS6 mabdetects a predominant 80 kDa glycoprotein with N-linkages. Minor sidebands are noted that may represent proteolytic degradations of the majorband or are side reactivites of the antibody (i.e., carbohydrate epitopeshared on more than one protein). No change in migration of the major 80kDa band is noted between reduced and non-reduced conditions signifyinga lack of interchain disulfide bonds (FIG. 3, B-C). Chloroformextraction studies are used to extract lipids from tissue sections priorto DS6 immunostaining and are then compared to DS6 immunostaining ontissue sections without prior chloroform extraction. While some generalreduction in staining intensity was noted in sections of fallopian tube,a more marked reduction was noted in the sections of ovarian tumors.Whether this apparent difference in efficiency of extraction in a normaltissue (i.e., fallopian tube) as compared to tumors is quantitative orqualitative is uncertain, however, ongoing studies into the chemicalnature of the epitope may help explain the significance of this finding.

The sialoglycotope nature of CA6 is similar to many of the histo-bloodgroup-related antigens such as sTn, sLea, sLex, some of which aredually-expressed as glycoproteins and glycolipids, however, unlike CA6,as glycoproteins, these are usually high molecular weight, carbohydraterich O-linked glycoproteins (mucins) (Magnani, J. L. et al., CancerRes., 43, 5489-5492 (1983); Muraro, R. et al., Cancer Res., 48,4588-4596 (1988)). The immunohistochemical distribution of thehisto-blood group related antigens, as reviewed by Zhang et al., wouldalso distinguish CA6 from the above sialyated histo-blood group antigens(Zhang et al., supra (1997)). The apical-staining pattern of DS6 on type2 pneumocytes, which lack sTn, sLea and sLex expression (Hachiya, T. etal., Virchows Arch., 434, 63-69 (1999); Atkinson, B. F. et al., CancerRes., 42, 4820-4823 (1982)), is strikingly similar to that reported forthe sTf (sialyl-Thomsen-Fridenreich) antigen (Hachiya et al., supra(1999)). However, if DS6 is reacting with the sTf epitope per se, itwould be expected to demonstrate a much broader range ofimmunoreactivity in human tissues than is seen in our studies, includingexpression in DS6-nonreactive epithelia, brain, lymphoid and othermesenchymal tissues (Cao et al., supra (1996)). The DS6 preabsorptionstudies using commercially available, highly purified sTn, sLea, Tn andTf carbohydrate antigens, in which the antigens failed to inhibit thetissue binding of DS6, also support CA6 being distinct from thesecarbohydrate antigens. Other TAAs to consider are those of approximately80 kDa such as OC133 (Berkowitz, R. et al., Am. J. Obstet. Gynecol.,146(6), 607-612 (1983); Masuho, Y. et al., Cancer Res., 44, 2813-2819(1984)), Ki-OCI-6-2 (Mettler, D. L. et al., Cancer, 65, 1525-1532(1990)), and the lactoferrin-related MAM5 (Zotter, S. et al., VirchowsArch. A. Pathol. Anat. Histopathol., 406(2), 237-251 (1985)), none ofwhich are expressed by fallopian tube, and 22-1-1, a mab to aneuraminidase resistant epitope on a 78 kDa protein, which is morecharacteristic of mucinous rather than serous ovarian tumors (Sonoda, K.et al., Cancer, 77(8), 1501-1509 (1996)).

Although the molecular weights of CA6 (80 kDa) and CA125 (>200 kDa) andtheir distribution in normal tissues differ (CA125 has been detected byIH in some normal epithelia of pancreas, ovary, fallopian tube, colon,gallbladder, stomach, endometrium, bronchus, lung, kidney and inmesothelium and amnion), the IH pattern of CA6 in our series of ovariantumors is similar to that reported for CA125 (Davis, H. M. et al.,Cancer Res., 46, 6143-6148, 1986; Nouwen, E. J. et al., Cancer Res.,46(2), 866-876 (1986); Dietel, M. et al., J. Cancer Res. Clin. Oncol.,111, 257-256 (1986); Itahashi, K. et al., Arch. Gyn. Obst., 243(3),145-155 (1988)). Both antigens are more characteristic of ovarian serouscarcinomas than ovarian mucinous neoplasms and both have a variegateddistribution pattern, often with luminal accentuation (Mattes, M. etal., Cancer Res. (suppl.), 50, 880-884 (1990)). In IH studies, bothantigens show a spectrum of expression ranging from strong and diffusein some tumors to patchy and variegated patterns in other neoplasms,with strongly positive groups of cells adjacent to nonreactive foci. Thedouble-label, two-color, sequential IH studies comparing CA6 to CA125demonstrate that mabs DS6 and OC125, while showing extensive overlap,also show areas in which only one of the antibodies is expressed,providing additional, complementary coverage of the tumor. Suchcomplementarity of TAA expression can have important clinicalimplications, whether for tailoring mab-based cancer therapies as“cocktails” matched to a given tumor's antigenic phenotype (Liao, S.- K.et al., Cancer Immunol. Immunother., 28, 77-86 (1989); Oldham, R. K.,Mol. Biother., 3, 148-161 (1991)), or for devising panels of tumormarkers to monitor tumor progression (Cane, P. et al., GynecologicOncol., 57, 240-245 (1995)).

In the sections of fallopian tubes, CA125 is distributed along luminalepithelial surfaces and is readily detected in the mesothelium.Conversely, CA6, which is more intensely expressed along the fallopiantube lumen, is not detected in the mesothelium. DS6 clearly recognizesan antigen that has a similar, but not identical, distribution to theCA125 antigen, with some distinct differences in patterns and sites ofantigen expression. The lack of CA6 expression in normal mesotheliumcould make DS6 a candidate for intraperitoneal antibody-targetedtherapeutic applications.

TABLE 1 Immunohistochemical detection of CA6 in tissue sections ofnormal adult tissues. Acetone-permeabilized and formalin-fixed,paraffin-embedded tissues were stained with mab DS6 as the primaryantibody by avidin-biotin immunohistochemistry. Formalin- Cryostatparaffin #positive/ #positive/ Tissue #tested #tested Positive celltypes/Comments Tissues with prominent, consistent CA6 expression withinand between specimens: Fallopian tube 3/3 6/6 (8/8)^(a) Luminal/apicalepithelial cell membrane Lung 2/2 5/5 Apical-aspect of type IIpneumocyte; occasional epithelial cell of bronchiole Urothelium 3/3 8/8Inner layers, luminal-facing aspect Tissues with low-level CA6expression, quite variable/focal within and between specimens: Pancreas2/2 5/6 Luminal-aspect of small to medium ducts Liver 1/2 3/5Luminal-aspect of occasional portal bile ducts Major salivary glandnt^(b) 2/3 Luminal-aspect of occasional medium-sized ducts Kidney 3/35/5 Luminal-aspect of scattered cells of distal nephron Endometrium,secretory 3/3 3/4 Focal luminal staining. Rare surface epithelial cell,2/4 Cervix 2/2 5/6 Focal, membrane rim pattern in metaplasticepithelium, transformation zone and 1/1 ectocervical epitheliumEndocervical glands 0/2^(c) 3/6 Pituitary 2/2 2/2 Focal staining of rimof follicles, anterior lobe Tissues lacking CA6 expression or rare focusonly: Cerebral cortex 0/3 0/3 Cerebellum 0/3 0/3 Spinal cord 0/3 0/3Large peripheral nerve 0/3 0/3 Trachea nt Parathyroid 0/2 1/4 Thyroid2/2 2/3 Rare apical-aspect of follicular epithelial cell Breast 1/3 2/4Rare apical-aspect of epithelial cell Adrenal 0/4^(d) 0/7 Esophagus 1/20/3 Single focus of suprabasal cell membrane staining, membrane rimpattern Stomach 0/4^(c) 0/6 [Intracellular canaliculas of parietal cellspositive, 3/4]^(e) Small intestine 0/4^(c) 0/7 Large intestine 0/4^(c)0/6 Gallbladder nt 0/3 Testes 0/4 0/5 [Weak positive rete testes,collecting ductules, 2/3; epididymis negative, 3] Prostate 0/1 0/4 [2specimens included periurethral prostatic ducts, weak positive] Ovary0/3 0/5 [one inclusion cyst positive, numerous inclusion cysts negative]Endometrium, proliferative 0/2 0/7 Heart 0/1 0/2 Skeletal muscle 0/2 0/3Stomach 0/4^(c) 0/6 [Intracellular canaliculas of parietal cellspositive, 3/4]^(e) Thymus nt 0/3 Spleen 0/2 0/3 Lymph node 0/2 0/3Epidermis 0/2 0/5 Other non-epithelial see Tissues consistently negativein numerous comments sections: mesothelium, rbc, wbc, lymphoreticularcells, fat, smooth muscle, nerve, endothelium Placenta nt 0/5 [Focalsquamous metaplastic amnion cell, rim pattern, 3/3] Chorion ntFootnotes: ^(a)( ) total number inclusive of double label studies;^(b)not tested; ^(c)staining of wispy extracellular material;^(d)unidentified spindle cells in capsule focally positive; ^(e)[ ]additional specificity not counted in table.

TABLE 2 Immunohistochemical detection of CA6 in gynecologic and relatedneoplasms/hyperplasias. Formalin-fixed, paraffin-embedded tissuesections were stained with mab DS6 as the primary antibody byavidin-biotin immunohistochemistry. Tissue #positive/#tested Ovarian:Serous cystadenoma 3/3 Serous borderline tumor 5/5 Serous adenocarcinoma11/11(18/18)^(a) Mucinous cystadenoma 0/2 Mucinous borderline tumor 0/2Mucinous adenocarcinoma 3/9 Endometroid, borderline 0/1 Endometrial:Clear cell adenocarcinoma Papillary serous adenocarcinoma 3/3Endometroid adenocarcinoma 1/1 Benign polyp 0/1 Peritoneum: Mesothelioma1/6 Reactive mesothelial hyperplasia 0/1 Serous surface carcinoma ofperitoneum 2/2 (3/3)  Footnotes: ^(a)( ) total number inclusive ofdouble label studies

TABLE 3 Expression of Tumor-Associated Antigen CA6 in GynecologicalNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive Nonreactive 1+ 2+ 3+ Cases/Total (Cases with <1% (Cases with1-9% (Cases with 10-49% (Cases with >50% Major Number neoplasticneoplastic neoplastic neoplastic Patterns of Neoplasm of Cases cellsreactive) cells reactive) cells reactive) cells reactive) ReactivityUterine Cervix Squamous Cell Carcinoma 10/14 4 4 3 3 Me, Cy SquamousDysplasia 0/7 7 Endocervical 0/2 2 Adenocarcinoma in Situ Endocervical2/3 1 1 1 Lu, Me Adenocarcinoma Adenosquamous Carcinoma 2/2 2 Me, CyEndometrium Papillary Serous 6/6 2 2 2 Lu, Me Adenocarcinoma Endometriod14/16 2 7 4 3 Lu, Me Adenocarcinoma Clear Cell Carcinoma  8/10 2 1 7 Lu,Me, Cy Mucinous Adenocarcinoma 3/5 2 1 2 Me, Cy Malignant MixedMullerian 10/18 8 5 1 4 Me, Cy Tumor, carcinomatous componentChoriocarcinoma 0/1 1 Molar Pregnancy 0/3 3 Uterus Low Grade Endometrial0/3 3 Stroma Sarcoma Undifferentiated Endometrial 0/1 1 SarcomaLeiomyoma 0/7 7 Leimyosarcoma 0/6 6 Ovary Serous Cystadenoma  9/12 3 1 8Lu Papillary Serous Borderline 10/10 1 2 7 Lu Tumor Papillary Serous56/58 2 4 9 43 Lu, Me, Cy Adenocarcinoma Papillary Serous Carcinoma14/14 1 13 Lu, Me, Cy Metastases Mucinous Cystadenoma  0/10 10 MucinousBorderline Tumor, 0/8 8 Intestinal Mucinous Borderline Tumor, 2/2 1 1 LuMullerian Mucinous Adenocarcinoma  0/10 10 Clear Cell Carcinoma 1/8 7 1Me Endometriod 5/8 3 3 1 1 Lu, Me Adenocarcinoma Benign Brenner Tumor6/9 3 2 1 3 Me, Lu, Cy Borderline Brenner Tumor 1/1 1 Me, Cy GranulosaCell Tumor  0/15 15 Fibroma/Thecoma  0/11 11 Sertoli Cell Tumor 0/1 1Yolk Sac Tumor 0/2 2 Struma Ovarii 0/1 1 Dysgerminoma 0/3 2 VulvaSquamous Cell Carcinoma 2/3 1 2 Me, Cy Hidradenoma Papilliferum 2/2 2 MePeritoneum Papillary Serous Surface 2/2 1 1 Lu, Me Carcinoma MalignantMesothelioma of 0/2 2 peritoneum Endosalpingiosis 1/1 1 Lu(nonneoplastic) Mesothelium (incidental 0/7 7 nonneoplastic)Abbreviations: Lu—Luminal portion of cell membrane; Me—Cell membrane;Cy—Cytoplasm; ICL—Intracytoplasmic lumina.

TABLE 4 Expression of Tumor-Associated Antigen CA6 in EpithelialNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive Cases/Total Nonreactive 1+ 2+ 3+ Major Number (Cases with <1%(Cases with 1-9% (Cases with 10-49% (Cases with >50% Patterns ofNeoplasm of Cases cells reactive) cells reactive) cells reactive) cellsreactive) CA6 Expression Breast Infiltrating Ductal Carcinoma 24/29 5 38 13 Lu, Me, ICL Infiltrating Lobular Carcinoma  5/11 6 1 1 3 ICLPleomorphic Lobular Carcinoma 1/2 1 1 ICL, Lu Colloid Carcinoma 6/7 1 33 Lu, Me Invasive Apocrine Carcinoma 1/1 1 ICL Medullary Carcinoma 1/1 1Lu, Me, ICL Tubular Carcinoma 1/1 1 Lu, ICL Invasive Papillary Carcinoma1/1 1 Lu, Me Brain Meningioma  3/25 22 2 1 Cy, Me Craniopharyngioma 1/21 1 Lu Gallbladder Gallbladder Adenocarcinoma 3/7 4 1 2 Lu, Me CysticDuct Adenocarcinoma 1/2 1 1 Lu Gastrointestinal Colonic Adenocarcinoma 3/21 18 2 1 Lu, Me Metastatic Colon Adenocarcinoma 0/3 3 Mucinous ColonAdenocarcinoma 0/6 6 Colonic Tubular Adenoma 0/1 1 Cloacogenic Carcinoma1/3 2 1 Lu, Me Duodenal Adenocarcinoma 0/1 1 Esophageal Adenocarcinoma1/4 3 1 Lu Esophageal Squamous Carcinoma 2/9 7 1 1 Me, Cy GastricAdenocarcinoma  7/19 12 2 5 Lu, Me Gastric Squamous Carcinoma 1/1 1 Me,Cy Kidney Clear Cell Carcinoma 13/18 5 13 Me, Lu Papillary Carcinoma 3/52 1 1 1 Lu, Me Granular Carcinoma 5/8 3 2 3 Me Chromophobe Carcinoma 3/41 2 1 Lu, Me, Cy Oncocytoma 2/2 1 1 Lu, Me, Cy Cortical Adenoma 1/1 1Lu, Me Wilms Tumor 1/9 8 1 Lu in tubules Larynx Squamous Cell Carcinoma10/27 17 8 1 1 Me, Cy Squamous Papilloma 1/1 1 Lu, Me LiverHepatocellular Carcinoma 1/8 7 1 Lu, Me, Cy Lung Adenocarcinoma  8/12 44 1 3 Lu, Me Squamous Cell Carcinoma  6/15 9 1 4 1 Me, Cy AdenosquamousCarcinoma 1/1 1 Me Pancreas Adenocarcinoma 8/9 1 2 3 3 Lu, Me PleuraMalignant Mesothelioma 0/6 6 Me Prostate Acinar Adenocarcinoma  4/13 9 31 Lu, Me Prostatic Duct Adenocarcinoma 0/5 5 Salivary Gland EpithelialNeoplasms [See Fig.] 21/90 69 12 6 3 Skin Epithelial Neoplasms [SeeFig.] 19/79 60 15 4 Testicle Teratoma, epithelial elements 2/5 3 2 LuThyroid Follicular Adenoma 0/2 2 Follicular Carcinoma  1/17 16 1 LuHurthle Cell Adenoma 2/5 3 1 1 Lu Hurthle Cell Carcinoma 2/5 3 1 1 LuHyperplastic Hurthle Cell Nodule 1/1 1 Lu Insular Carcinoma 1/1 1 Lu, MePapillary Carcinoma 16/27 11 9 7 Lu, Me Thymus Thymoma  0/17 17 Lu, MeThymic Carcinoma 0/1 1 Squamous Cell Carcinoma 1/1 1 Lu, Me UrinaryBladder Transistional Cell Carcinoma 18/21 3 8 5 5 Lu, Me Squamous CellCarcinoma 0/1 1 Abbreviations: Lu—Luminal portion of cell membrane;Me—Cell membrane; Cy—Cytoplasm; ICL—Intracytoplasmic lumina.

TABLE 5 Expression of Tumor-Associated Antigen CA6 in Skin Neoplasms asImmunohistochemically Detected by Monoclonal Antibody DS6 ReactiveCases/Total Nonreactive 1+ 2+ 3+ Major Number (Cases with <1% (Caseswith 1-9% (Cases with 10-49% (Cases with >50% Patterns of Neoplasm ofCases cells reactive) cells reactive) cells reactive) cells reactive)CA6 Expression Basal Cell Carcinoma  0/19 19 Chondroid Syringoma 0/1 1Cylindroma 0/3 3 Dermatofibroma 0/9 9 Dermatofibosarcoma 0/3 3Protuberans Eccrine Acrospiroma 11/13 2 10 1 Me, Lu Eccrine Spiradenoma1/7 6 1 Lu Eccrine Carcinoma 0/1 1 Hemangioma 0/8 8 Leiomyoma 0/8 8Leiomyosarcoma 0/1 1 Merkel Cell Carcinoma 1/2 1 Punctate Cy, MeNeurofibroma  0/13 13 Nevus Sebaceous 0/5 5 Pilomatricoma 0/1 1 Poroma3/6 3 2 1 Me, Lu Seborrheic Keratosis 0/1 1 Spiradenoma 1/7 6 1 LuSyringoma 0/2 2 Syringocystadenoma 3/3 1 2 Lu PapilliferumTrichepithelioma  0/10 10 Urticaria Pigmentosa 0/3 3 Xanthogranuloma 0/44 Angiofibroma 0/2 2 Angiosarcoma 0/2 2 Glomangioma 0/1 1Hemangiopericytome 0/2 2 Malignant 0/1 1 Hemangiopericytoma Blue Nevus0/5 5 Spitz Nevus 0/2 2 Malignant Melanoma  0/10 10 Abbreviations:Lu—Luminal portion of cell membrane; Me—Cell Membrane; Cy—Cytoplasm.

TABLE 6 Expression of Tumor-Associated Antigen CA6 in Salivary GlandNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive Cases/Total Nonreactive 1+ 2+ 3+ Major Number (Cases with <1%(Cases with 1-9% (Cases with 10-49% (Cases with >50% Patterns ofNeoplasm of Cases cells reactive) cells reactive) cells reactive) cellsreactive) CA6 Expression Acinic Cell Carcinoma 0/8 8 Adenoid CysticCarcinoma  7/14 7 5 1 1 Lu Basal Cell Adenoma 1/4 3 1 Lu Basal CellCarcinoma 0/1 1 Clear Cell Carcinoma 0/5 5 Canalicular Adenoma 0/1 1Malignant Mixed Tumor 0/1 1 Mucoepidermoid Carcinoma 3/4 1 3 Lu, MePleomorphic Adenoma  4/30 26 4 Lu Polymorphous Low Grade 0/1 1 CarcinomaSalivary Duct Carcinoma 4/5 1 2 2 Lu, Me, Cy Warthin Tumor  2/16 14 2 LuAbbreviations: Lu—Luminal portion of cell membrane; Me—Cell Membrane;Cy—Cytoplasm.

TABLE 7 Non-Epithelial Neoplasms Which Do Not Express Tumor-AssociatedAntigen CA6 as Studied Immunohistochemically with Monoclonal AntibodyDS6 TOTAL CASES SITE NEOPLASM STUDIED Adrenal Neuroblastoma &Ganglioneuroblastoma 21 Pheochromocytoma 4 Breast Fibroadenoma 1 BrainGlioblastoma Multiforme 40 Anaplastic Astrocytoma 5 Oligodendroglioma 9Pilocytic Astrocytoma 12 Medulloblastoma 10 Ependymoma 15 Ganglion CellTumors 4 Central Neurocytoma 1 Schwannoma 29 Hemanioblastoma 6Gastrointestinal Appendiceal Carcinoid Tumor 5 AppendicealAdenocarcinoid 1 Colonic Carcinoid Tumor 5 Gastric Carcinoid 6 GIMesenchymal Tumors 17 Lung Carcinoid Tumor 21 Small Cell Carcinoma 5Large Cell Neuroendocrine Carcinoma 6 Lymphoma Diffuse Small CellLymphomas 31 Large Cell Lymphomas 19 Follicular Lymphomas 36 BlasticLymphomas 11 Hodgkins Disease 29 Multiple Myeloma 23 Langerhans CellHistiocytosis 8 Leukemias Acute Lymphoblastic Leukemia 4 AcuteMyelogenous Leukemia 5 Chronic Myelogenous Leukemia 5 Oral Ameloblastoma6 Tissues Pancreas Islet Cell Tumor 6 Parathyroid Adenoma 8 Skin BlueNevus 5 Spitz Nevus 2 Skin Malignant Melanoma 10 Dermatofibroma 9Dermatofibosarcoma Protuberans 4 Hemangioma 8 Leiomyoma 8 Leiomyosarcoma1 Neurofibroma 13 Urticaria Pigmentosa 3 Xanthogranuloma 4 Angiofibroma2 Angiosarcoma 2 Glomangioma 1 Hemangiopericytoma 2 MalignantHemangiopericytoma 1 Soft Tissue Angiosarcoma 1 Alveolar Soft PartsSarcoma 1 Chondrosarcoma 2 Fibrosarcoma 2 Granular Cell Tumor 7Hemangiopericytoma 1 Leiomyosarcoma 14 Liposarcoma 4 Malignant FibrousHistiocytoma 6 Malignant Schwannoma 6 Neurofibroma 1 Osteosarcoma 2Rhabdomyosarcoma 3 Undifferentiated Sarcoma 2 Paraganglioma 5 PrimitiveNeuroectodermal Tumor 3 Testicle Choriocarcinoma 4 Embryonal Carcinoma16 Seminoma 18 Teratoma, Non-epithelial elements 5 Yolk Sac Tumor 12Thyroid Medullary Carcinoma 3

TABLE 8 Non-Epithelial Neoplasms Which Express Tumor-Associated AntigenCA6 as Detected Immunohistochemically by Monoclonal Antibody DS6Reactive Cases/Total Nonreactive 1+ 2+ 3+ Major Number (Cases with <1%(Cases with 1-9% (Cases with 10-49% (Cases with >50% Patterns ofNeoplasm of Cases cells reactive) cells reactive) cells reactive) cellsreactive) CA6 Expression Merkel Cell Carcinoma 1/2 1 1 Membranous &cytoplasmic Pituitary Adenoma  2/21 19 2 Luminal in pseudorosettes;punctate in cytoplasm Small Intestinal Carcinoid 1/9 8 1 Luminal inTumor rosettes Synovial Sarcoma 1/3 2 1 Luminal and membranous

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1.-22. (canceled)
 23. A method of imaging cancer in a subject,comprising administering to the subject a monoclonal antibody selectedfrom the group consisting of a monoclonal antibody produced by thehybridoma cell line DS6 deposited with the American Type CultureCollection as deposit number PTA-4449 and fragments of the foregoingwhich specifically bind the epitope bound by a monoclonal antibodyproduced by the hybridoma cell line DS6 deposited with the American TypeCulture Collection as deposit number PTA-4449, wherein the monoclonalantibody is detectably labeled with a radioisotope.
 24. The method ofclaim 23, wherein the radioisotope is derived from iodine, yttrium,astatine, bismuth, copper, rhenium or lead.
 25. The method of claim 23,wherein the radioisotope is derived from iodine.
 26. The method of claim23, wherein the radioisotope is derived from yttrium.
 27. The method ofclaim 23, wherein the radioisotope is derived from astatine.
 28. Themethod of claim 23, wherein the radioisotope is derived from bismuth.29. The method of claim 23, wherein the radioisotope is derived fromcopper.
 30. The method of claim 23, wherein the radioisotope is derivedfrom rhenium.
 31. The method of claim 23, wherein the radioisotope isderived from lead.
 32. The method of claim 23, wherein the radioisotopeis ¹³¹I, ⁹⁰Y, ²¹¹At, ²¹²Bi, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re or ²¹²Pb.
 33. The methodof claim 23, wherein the monoclonal antibody is a chimeric antibody. 34.The method of claim 23, wherein the monoclonal antibody is a humanizedantibody.
 35. The method of claim 23, wherein the cancer is ovarian,endometrial, breast, pancreatic, lung or urinary bladder cancer.
 36. Themethod of claim 23, wherein the cancer is ovarian cancer.
 37. The methodof claim 23, wherein the cancer is endometrial cancer.
 38. The method ofclaim 23, wherein the cancer is breast cancer.
 39. The method of claim23, wherein the cancer is pancreatic cancer.
 40. The method of claim 23,wherein the cancer is lung cancer.
 41. The method of claim 23, whereinthe cancer is urinary bladder cancer.